Cyanotryptophans as Novel Fluorescent Probes for Studying Protein Conformational Changes and DNA-Protein Interaction

Poulami Talukder, Shengxi Chen, Basab Roy, Petro Yakovchuk, Michelle M. Spiering, Mohammad P. Alam, Manikandadas M. Madathil, Chandrabali Bhattacharya, Stephen J. Benkovic, Sidney Hecht

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

Described herein are the syntheses and photophysical characterization of three novel cyanotryptophans, and their efficient incorporation into proteins as fluorescent probes. Photophysical characteristics indicated that each was significantly brighter and red-shifted in fluorescence emission relative to tryptophan. Each analogue was used to activate a suppressor tRNA transcript and was incorporated with good efficiency into two different positions (Trp22 and Trp74) of Escherichia coli dihydrofolate reductase (ecDHFR). The Trp analogues could be monitored selectively in the presence of multiple native Trp residues in DHFR. 6-CNTrp (A) formed an efficient Förster resonance energy transfer (FRET) pair with l-(7-hydroxycoumarin-4-yl)ethylglycine (HCO, D) at position 17. Further, 6-CNTrp (A) was incorporated into two DNA binding proteins, including the Klenow fragment of DNA polymerase I and an RNA recognition motif (RRM2) of heterogeneous nuclear ribonucleoprotein L-like (hnRNP LL). Using these proteins, we demonstrated the use of FRET involving A as a fluorescence donor and benzo[g]quinazoline-2,4-(1H,3H)-dione 2′-deoxyriboside (Tf) or 4-aminobenzo[g]quinazoline-2-one 2′-deoxyriboside (Cf) as fluorescent acceptors to study the binding interaction of the Klenow fragment with duplex DNA oligomers (labeled with Tf), or the domain-specific association between hnRNP LL and the BCL2 i-motif DNA (labeled with Cf). Thus, the non-natural amino acid could be used as a FRET partner for studying protein-nucleic acid interactions. Together, these findings demonstrate the potential utility of 6-CNTrp (A) as a fluorescence donor for the study of protein conformational events.

Original languageEnglish (US)
Pages (from-to)7457-7469
Number of pages13
JournalBiochemistry
Volume54
Issue number51
DOIs
StatePublished - Dec 29 2015

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of 'Cyanotryptophans as Novel Fluorescent Probes for Studying Protein Conformational Changes and DNA-Protein Interaction'. Together they form a unique fingerprint.

  • Cite this

    Talukder, P., Chen, S., Roy, B., Yakovchuk, P., Spiering, M. M., Alam, M. P., Madathil, M. M., Bhattacharya, C., Benkovic, S. J., & Hecht, S. (2015). Cyanotryptophans as Novel Fluorescent Probes for Studying Protein Conformational Changes and DNA-Protein Interaction. Biochemistry, 54(51), 7457-7469. https://doi.org/10.1021/acs.biochem.5b01085